人源中和性抗高致病性禽流感病毒H5N1基因工程全抗体的研制Generation of Neutralizing Recombinant Human Antibodies for Targeting Highly Pathogenic Avian Influenza A(H5N1) Virus
孙丽娜,刘琴芝,王敏,李川,李梓,胡晓芬,朱莉莉,李群,王世文,舒跃龙,梁米芳,李德新
摘要(Abstract):
运用噬菌体表面呈现技术,从禽流感病人恢复期血中获得淋巴细胞,通过基因工程手段,构建了人源抗H5N1禽流感病毒基因工程抗体文库。用纯化的人源H5N1禽流感病毒颗粒(A/Anhui/1/2005)及重组血凝素蛋白HA(A/VietNam/1203/2004)对Fab噬菌体抗体库进行富集筛选,成功地获得了抗禽流感病毒H5N1血凝素蛋白HA的人源单抗Fab段基因,并在大肠杆菌中获得有效表达。通过序列测定确定抗体轻重链型别,然后将阳性克隆的轻链和重链Fd段基因分别克隆入全抗体表达载体pAC-L-Fc后转染昆虫Sf9细胞,利用杆状病毒/昆虫细胞系统实现全抗体的分泌型表达。用ELISA、IFA和流式细胞术对所获人源单抗的功能特性进行鉴定。结果表明,我们获得了2株特异性针对H5N1禽流感病毒血凝素蛋白HA而与甲1型和甲3型人流感病毒无交叉反应的人源单抗(AVFluIgG01、AVFluIgG03)。微量中和试验结果表明,除A/Guangdong/1/2006外,AVFlu-IgG01能够广泛地中和HA基因进化上属于Clade2的中国南方、北方及中部地区的H5N1禽流感病毒分离株,同时还对属于CladeⅠ的越南H5N1分离株A/VietNam/1203/2004具有中和活性;AVFluIgG03虽然不能中和A/VietNam/1203/2004,但是对属于Clade2的所有中国H5N1分离株均具有中和作用。人源中和性抗禽流感病毒H5N1基因工程全抗体的获得不仅为高致病性禽流感病毒H5N1的预防和治疗带来了希望,同时也为其疫苗研制提供了新的思路。
关键词(KeyWords): 禽流感病毒;噬菌体表面呈现;基因工程抗体;Fab抗体
基金项目(Foundation): 国家863计划(2006AA02A252);; 国家科技支撑计划(2006BAD06A15)
作者(Author): 孙丽娜,刘琴芝,王敏,李川,李梓,胡晓芬,朱莉莉,李群,王世文,舒跃龙,梁米芳,李德新
参考文献(References):
- [1]World Health Organization(WHO)[DB].http://www.who.int/csr/disease/avian-influenza/country/ca-ses-table-2008-02-21/en/index.ht ml
- [2]Wiley D C,Skehel J J.The structure andfunction of the hemagglutinin membrane glycoprotein of influenza virus[J].Annu Rev Biochem,1987,56:365-394.
- [3]Skehel J J,Wiley D C.Receptor binding and membrane fusionin virus entry:the influenza hemagglutinin[J].Annu Rev Biochem,2000,69:531-569.
- [4]Stephenson I,Nicholson K G,Wood J M,et al.Con-fronting the avian influenza threat:vaccine development for a potential pandemic[J].Lancet Infect Dis,2004,4:499-509.
- [5]de Jong MD,Tran T T,Truong H K,et al.Oseltami-vir resistance during treat ment of influenza A(H5N1)infection[J].N Engl J Med,2005,353:2667-2672.
- [6]Si mmons P C,Bernasconi N L,Suguitan A L,et al.Prophylactic and therapeutic efficacy of human mono-clonal antibodies against H5N1influenza[J].Plos Med,2007,4(5):0928-0936.
- [7]Hanson B J,Boon A C,Li m A P,et al.Passive i mmu-noprophylaxis and therapy with humanized monoclonal antibody specific for influenza A H5hemagglutinin in mice[J].Respir Res,2006,7:126-127.
- [8]Barbas C FⅢ,Kang A S,larner R A.Assembly of combinatorial antibody libraries on phage surface:the geneⅢsite[J].Proc Natl Acad Sci USA,1991,88(18):7978-7982.
- [9]World Health Organization(WHO)[DB].WHO manual on ani mal influenza diagnosis and surveillance.http://www.who.int/vaccine-research/diseases/influenza/WHO-manual-on-ani mal-diagnosis-and-surveillance-2002-5.pdf
- [10]郭元吉,程晓雯.流行性感冒病毒及其试验技术[M].北京:中国三峡出版社,1997:94-96.
- [11]Liang M F,Stefan D,Li D X,et al.Baculovirus ex-pression cassette vectors for rapid production of com-plete human IgG from phage display selected antibody fragments[J].J I mmunol Meth,2001,247:119-130.
- [12]Harlow E,Lane D.Antibodies:A Laboratory Manual[M].New York:Cold Spring Harbor Laboratory Press,1988.
- [13]Heitner T,Moor A,Jennifer L,et al.Selection of cell binding and internalizing epidermal growth factor re-ceptor antibodies froma phage library[J].J I mmunol Meth,2001,248:17-30.
- [14]World Health Organization(WHO).Manual on Influ-enza Microneutralization Assay[M].CDC Influenza Training Course,2004.
- [15]Daniels R S,Jeffries S,Yates P,et al.The receptor-binding and membrane-fusion properties of influenza vi-rus variants selected using anti-haemagglutinin mono-clonal antibodies[J].EMBOJ,1987,6:1459-1465.
- [16]舒跃龙,蓝雨,温乐英,等.我国分离人H5N1禽流感病毒血凝素基因特性的研究[J].中华实验和临床病毒学杂志,2006,20:8-10.